The molecular structure of nucleic acids provides for specific detection by means of complementary base pairing of oligonucleotide probes or primers to sequences that are unique to specific target organisms or tissues. Since all biological organisms or specimens contain nucleic acid of specific and defined sequences, a universal strategy for nucleic acid detection has extremely broad applications in a number of diverse research and development areas as well as commercial industries. The potential for practical uses of nucleic acid detection was greatly enhanced by the description of methods to amplify or copy, with fidelity, precise sequences of nucleic acid found at low concentration to much higher copy numbers, so that they are more readily observed by detection methods.
The original amplification method is the polymerase chain reaction described by Mullis et al. (U.S. Pat. No. 4,683,195, U.S. Pat. No. 4,683,202, and U.S. Pat. No. 4,965,188, all of which are specifically incorporated herein by reference). Subsequent to the introduction of PCR, a wide array of strategies for amplification have been described. See, for example, U.S. Pat. No. 5,130,238 to Malek, nucleic acid sequence based amplification (NASBA); U.S. Pat. No. 5,354,668 to Auerbach, isothermal methodology; U.S. Pat. No. 5,427,930 to Buirkenmeyer, ligase chain reaction; and, U.S. Pat. No. 5,455,166 to Walker, strand displacement amplification (SDA); all of which are specifically incorporated herein by reference. Some of these amplification strategies, such as SDA or NASBA, require a single stranded nucleic acid target. The target is commonly rendered single stranded via a melting procedure using high temperature prior to amplification. The instant invention provides a novel mechanism for converting double stranded nucleic acid to single stranded nucleic acid without that conventional melting step.
Prior to nucleic acid amplification and detection, the target nucleic acid must be extracted and purified from the biological specimen such that inhibitors of amplification reaction enzymes are removed. Further, a nucleic acid target that is freely and consistently available for primer annealing must be provided. A wide variety of strategies for nucleic acid purification are known. These include, for example, phenol-chloroform and/or ethanol precipitation (Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.), high salt precipitation (Dykes (1988) Electrophoresis 9:359-368), proteinase K digestion (Grimberg et al. (1989) Nucleic Acids Res 22:8390), chelex and other boiling methods (Walsh et al. (1991) Bio/techniques 10:506-513) and solid phase binding and elution (Vogelstein and Gillespie (1979) Proc. Nat. Acad. Sci. USA 76:615-619), all of which are specifically incorporated herein by reference.
The analysis of nucleic acid targets therefore consists of three steps: nucleic acid extraction/purification from biological specimens, direct probe hybridization and/or amplification of the specific target sequence, and specific detection thereof. In currently employed conventional protocols each of these three steps is performed separately, making nucleic acid analysis labor intensive. Further, numerous manipulations, instruments and reagents are necessary to perform each step of the analysis.
Another concern with current methodologies is the significant chance of specimen cross contamination; between concurrently run specimens or from a previously amplified sample. It would be advantageous to eliminate the melt step necessary for generating single strand nucleic acid for probe hybridization or amplification primer annealing, and directly integrate the three nucleic acid analysis steps so as to simplify the analysis procedure and methodologies, as well as reduce and/or remove the risk of cross contamination. The invention discussed herein provides a method for a direct interface of the extraction and hybridization or amplification steps discussed above.
For analysis purposes, nucleic acid must frequently be extracted from extremely small specimens in which it is difficult, if not impossible, to obtain a second confirmatory specimen. Examples include analysis of crime scene evidence or fine needle biopsies for clinical testing. In such examples, the extent of the genetic testing and confirmation through replica testing is, thus, limited by the nucleic acid specimen size. Using conventional extraction protocols for these small specimens, the nucleic acid is often lost or yields are such that only a single or few amplification analyses are possible. The present invention provides a method for irreversibly binding and thus, permanently archiving, nucleic acid from specimens. That is to say, the nucleic acid is neither altered nor exhausted during analysis, and therefore, is able to be reanalyzed an unlimited number of times. This invention takes advantage of solid phase DNA binding properties known but believed by the skilled artisan to be incompatible with nucleic acid analysis. In addition, binding properties of use for RNA analysis are characterized.
Specimens that contain high levels of endogenous or background nucleic acid such as blood are extremely difficult to analyze for the presence of low level specific targets. Solid phases with high nucleic acid avidity can be utilized to irreversibly capture oligonucleotide or probe sequences. By changing buffer conditions these materials can then selectively capture target sequences even in the presence of high levels of background nucleic acid.
The requirements for binding of DNA to solid phases and subsequently being able to elute them therefrom have been described by Boom (U.S. Pat. No. 5,234,809, specifically incorporated herein by reference) and Woodard (U.S. Pat. No. 5,405,951, U.S. Pat. No. 5,438,129, U.S. Pat. No. 5,438,127, all of which are specifically incorporated herein by reference). Specifically, DNA binds to solid phases that are electropositive and hydrophilic. Solid phase materials consisting of the atoms Silicon (Si), Boron (B), or Aluminum (Al) can be rendered sufficiently hydrophilic by hydroxyl (--OH) or other groups to result in a surface that irreversibly binds DNA, while proteins or inhibitors do not bind. The binding of RNA has not been previously characterized and is revealed in the present invention. Since conventional purification methods require elution of the bound nucleic acid, these solid phase materials are described as being of no use for DNA purification. In fact, considerable effort has been expended to derive solid phase materials sufficiently electropositive and hydrophilic to adequately bind nucleic acid and yet allow for its elution therefrom. (See, for example, U.S. Pat. Nos. 5,523,392, 5,525,319 and 5,503,816 all to Woodard, and all of which are specifically incorporated herein by reference). The present invention uses solid phase matrixes to irreversibly bind nucleic acid and teaches methods for direct solid phase nucleic acid manipulation, hybridization, and/or amplification. That is, analysis is performed without elution of the nucleic acid from the solid phase.
Boom, supra, describes solid phase DNA amplification using high chaotropic salt to reversibly bind to silica. When this solid phase is placed in the amplification reaction buffer, the nucleic acid is, in fact, eluted. Therefore, the amplification actually occurs in solution, not on solid phase. Furthermore, since binding is not irreversible, the amplification can only be performed once. Del Rio et al. ((1996) Bio/techniques 20:970-974) describe filter entrapment of nucleic acid in a manner allowing for repeat amplification. However, they do not describe a binding mechanism that is irreversible, therefore the method is only recommended for analysis of higher nucleic acid concentrations and then, only for a limited number of analyses.
The instant invention is directed to a novel method for converting double stranded nucleic acid to single stranded nucleic acid without any melting step and provides methods for rapid DNA and RNA capture that directly interface extraction and purification with either hybridization and/or amplification. The present invention further provides a method for irreversibly binding, and thus, permanently archiving nucleic acid from specimens. The present invention uses solid phase matrixes to irreversibly bind nucleic acid and teaches true, direct solid phase manipulation and analyses including enzyme recognition, hybridization, and primer dependent amplification. True solid phase analysis provides for stringent aqueous washes, rapid automatable nucleic acid capture and purification, selective nucleic acid detection, repeat and/or expanded analysis of the bound nucleic acid, and long term storage of nucleic acid. Each of these disclosed methodologies overcomes the drawbacks of the prior art.